In the field of pathological diagnosis, it is useful to detect a specific cell(s) or tissue from a specimen of a sampled tissue section and quantify the expression level of a substance associated with a lesion on the specific cell(s) or tissue, and methods utilizing immunohistochemistry have been examined for this purpose.
Immunohistochemistry (IHC) is widely known as a histological (histochemical) technique for detecting an antigen in a tissue sample using an antibody. This immunohistochemistry is sometimes referred to as “immunostaining” or the like (hereinafter, the term “immunohistochemical staining” may also be used for immunohistochemistry) because it involves a color-developing operation in order to visualize an antigen-antibody reaction that is intrinsically invisible. Due to this characteristic feature of visualizing the location of an antigen-antibody reaction, immunohistochemistry is widely used in the fields of medicine and life chemistry for the purpose of detecting the location of a biological substance in a tissue sample.
In IHC, as a method of visualizing the location of an antigen-antibody reaction, a staining method that can be observed in a bright field is widely used and, specifically, a technique using a substrate that is converted into a pigmentary substance by an enzyme is commonly used. For example, in clinical scenes, it is widely performed to stain and thus visualize an antibody bound to an antigen of interest in a tissue sample using peroxidase (POD) and diaminobenzidine (DAB) and to detect the expression amount of the specific antigen by bright-field observation through the thus visualized antibody. This bright-field observation is advantageous in that, as compared to the below-described method using a light-emitting substance, more detailed information on a target molecule can be obtained by comprehensively judging the information obtained in an analog manner such as staining color. As a method of distinguishing cytoma and specifying the position of the cytoma in a specimen using such immunostaining, an immunostaining technology which uses a marker specifically expressed in cells as a target has been generally employed.
As a method of evaluating the expression level of a biological substance of interest in a tissue section with high accuracy using immunohistochemistry, there is known a method which comprises performing immunostaining with a fluorescent substance-containing nanoparticle, detecting the thus generated fluorescent bright dots and then evaluating the expression level of a biological substance (Patent Document 1). In this method, the brightness distribution corresponding to the biological substance of interest can be measured by the use of the fluorescent substance-containing nanoparticle; however, it is difficult to specify the positional relationship between a specific tissue or cell in a tissue section and the biological substance of interest and thus to measure the brightness distribution by selecting the biological substance of interest expressed on the specific tissue or cell.
In addition, a method of simultaneously performing bright-field observable staining by an enzyme antibody method (DAB staining that uses an enzyme reaction) and fluorescent dye staining by a fluorescent antibody method (staining that uses a fluorescent dye) on a single tissue section has been reported (Non-patent Documents 1 and 2). In this method, although a biological substance of interest is quantified using a fluorescent dye, since a sufficient fluorescence intensity cannot be obtained at those spots where the biological substance is expressed in a small amount, it is difficult to quantify the biological substance of interest in some cases. For instance, in an example in which Ki-67-positive cells were stained by an enzyme antibody method and cytokeratin was fluorescently stained by a fluorescent antibody method, it is reported that there were cases where fluorescence observation of cytokeratin was difficult due to inadequate amount of fluorescence (Non-patent Document 2).
As a method of quantifying a protein by multiple fluorescent staining, there has also been proposed a method that comprises staining a protein of interest to be quantified and a reference protein with different fluorescent dye-labeled antibodies, measuring the total fluorescence intensities of the respective proteins and then quantifying the protein of interest based on the ratio of the thus measured total fluorescence intensities (Patent Document 2).
As described above, with regard to a method of detecting a specific tissue or cell in a sampled tissue section and quantifying a biological substance of interest that is expressed on the specific tissue or cell, there is a demand for a method of more accurately specifying both the expression position (s) and the expression amount.